Authors: Aaryan Patel, Vania Carmona-Alcocer, Kelvin Vuu, Phillip Karpowicz
Circadian rhythms are internal mechanisms present in nearly all animals that time behaviour and physiology in 24-hour oscillation periods. We attempted to develop TaqMan Open Array assays to detect conserved regions of clock genes, initially targeting 13 circadian clock genes, and 3 control genes. Primers were designed for regions of homology in: 1) the GEN-FISH species of interest, 2) Salmonids, 3) the Chinook salmon (Onchorhychus tshawytscha), and following their design were tested using Chinook cDNA by SYBR Green qPCR. Only 5 targets produced qPCR amplicons for #1, however, it was possible to design primers targeting Salmonid cDNA for all genes. These were used to construct TaqMan Open Array chips.
To test whether these could detect circadian gene expression, we investigated Chinook muscle tissues in four environmental conditions. We examined Chinook salmon parr entrained in 4 different photoperiod conditions: 12-hour Light:12-hour Darkness (12:12 LD), 8:16 LD, 16:8 LD, and 24 DD (complete darkness). The final condition tests circadian rhythmicity which will persist in the absence of photoperiod. Universal primers fared extremely poorly in these experiments, only 1/5 genes were detected in a robust manner. However, all Chinook and Salmonid primers were detected apart from the genes Per1 and Per2. Unsurprisingly, circadian clock genes oscillated in a 24-hour manner in the 12:12 LD condition, but these are altered in 8:16 LD, and 16:8 LD, in a gene-specific manner. Importantly, these genes persisted in DD conditions, demonstrating that the circadian clock is functional and the gene expression observed is not due to stochastic changes. Our ongoing work will include deeper analysis on the changes in clock genes’ expression between these photoperiod conditions tested, and how these influence muscle tissue growth and morphology. Our data supports the use of TaqMan Open Array to detect circadian genes at the family level to test circadian health.
